One of the most common features that distinguishes cancer cells from normal cells is the presence of aneuploidy. We have recently discovered a cause of aneuploidy in a substantial fraction of malignant human tumors - deletions, somatic mutations, and loss of expression of the STAG2 gene (Science 336:1039-1043, 2011). The encoded STAG2 protein is a key component of the multi-protein cohesin complex which regulates sister chromatid cohesion and helps ensure faithful chromosome segregation during mitosis. In this grant we propose a multi-faceted effort to further define the mechanism through which STAG2 inactivation leads to chromosomal instability, aneuploidy, and cancer. In Aim #1 we will evaluate the effects of tumor-derived mutations in STAG2 on sister chromatid cohesion and protein-protein interactions. The goal is to functionally evaluate mutations in STAG2 to reveal the key activities of STAG2 that are lost during tumorigenesis. In Aim #2 we will determine the effect of STAG2 truncating mutations on the protein composition of cohesin and its ability to interact with chromatin at different phases of the cell cycle. These experiments wil enable us to identify key mechanisms by which STAG2 inactivation leads to chromosomal instability and aneuploidy. In Aim #3 we will determine the role of STAG2 inactivation on the initiation and maintenance of transformation in human astrocytes and GBM cells. These experiments will enable us to determine if STAG2 inactivation results in enhanced susceptibility to malignant transformation. In Aim #4 we will determine if mutations in STAG2 cause GBM cells to be sensitized to ionizing radiation and DNA damaging chemotherapeutic drugs. The broad goal of the proposed research program is to determine the mechanism through which STAG2 inactivation leads to chromosomal instability, aneuploidy, and cellular transformation, making it possible to develop strategies for identifying novel anticancer therapeutics that specifically target aneuploid cells.